This proposal will continue our studies on the structure and function of the neuronal intermediate filaments. Although our focus had been primarily on the neurofilament triplet proteins, NF-L, NF-M and NF-H, the last grant period has also yielded a wealth of data on alpha-internexin, another neuronal intermediate filament protein, first described in our laboratory. Over this time, we obtained cDNA and genomic clones, as well as monoclonal and polyclonal antibodies, and studied the in vivo and in vitro assembly of these neuronal intermediate filament proteins. We have shown that alpha-internexin is the first neuronal intermediate filament protein to be expressed by most if not all neurons during development. The first specific aim is to compare the importance of different structural domains of the neuronal intermediate filament proteins in in vivo and in vitro assembly. These studies will enable us to understand the mechanisms by which intermediate filaments are formed in neurons and should begin to explain the importance of the diversity of neuronal intermediate filaments. The second aim is to determine the control elements responsible for the tissue-specific expression of alpha- internexin. This aim will allow us to determine how this neuron specific gene is regulated. It could also provide us with the tools necessary to target proteins specifically to the nervous system. The third aim is to accomplish the targeted disruption of the gene for alpha-internexin in transgenic mice. these experiments should help us in determining the function of this protein. The roles that neuronal intermediate filaments play in development, axonal integrity and neurological diseases are not clear, although they are implicated in all of them. Recent studies on the genetic skin diseases, Epidermolysis Bullosa Simplex and Epidermolytic Hyperkeratosis point to non-life threatening, but still debilitating illnesses resulting from mutations in the keratins. Similarly, neuronal intermediate filaments may not be essential for survival per se, but defects in neuronal intermediate filaments are likely to be related to neuronal diseases. In this proposal, we hope to determine the function of one of the neuronal intermediate filament proteins, alpha-internexin and identify its role in the nervous system and in neurological diseases.